Chemical priming potentiates mesothelin-targeting chimeric antigen receptor-engineered NK-92 antitumor activity by improving tumor trafficking and cytotoxic killing dynamics.

Introduction: Chimeric antigen receptor-engineered natural killer (CAR-NK) cells have emerged as a promising strategy for cancer immunotherapy; however, their efficacy against solid tumors remains limited by inefficient tumor trafficking and impaired cytotoxic function within the tumor microenvironment. Here, we investigated whether a non-genetic chemical priming strategy could pre-arm CAR-NK cells and enhance their migratory and cytotoxic functions. Methods: Based on our previous findings that transient exposure to 25 kDa branched polyethylenimine (25KbPEI) induces a primed phenotype, mesothelin-targeting CAR-NK-92 cells were chemically primed and evaluated for migration, cytotoxicity, killing dynamics, perforin accumulation, cytokine production, and in vivo antitumor efficacy in a SKOV3 xenograft model. Results: Chemical priming significantly enhanced cytotoxicity and degranulation against ovarian cancer cells without compromising cell viability. Primed CAR-NK cells showed increased CCR7 expression and improved tumor-directed migration. Live-cell imaging further revealed accelerated target engagement and shortened killing time, indicating enhanced cytotoxic kinetics. In addition, chemical priming increased perforin accumulation and IFN-γ production. In the SKOV3 xenograft model, primed CAR-NK cells achieved superior tumor control and increased intratumoral infiltration compared with non-primed CAR-NK cells, while maintaining a favorable safety profile. Discussion: Collectively, these findings demonstrate that chemical priming enhances CAR-NK cell function and provides a promising non-genetic strategy to improve CAR-NK cell activity against solid tumor models.